Ligation hydrazone

The aromatic nature of the SFB and SANH reactants create a hydrazone structure with unique absorptivity for monitoring the course of the ligation process. The aromatic hydra-zone bond has a maximal absorbance at 354nm and a molar extinction coefficient equal to... 

Once a molecule is modified with a hydrazine reagent and another molecule is modified with the benzaldehyde compound, they may be combined to form the final conjugate, which will result in a hydrazone linkage between the two molecules. In addition, chemoselective ligation using aldehyde/hydrazine reactions may be done to immobilize biomolecules. In this regard, one modified component may be a surface and the other one an antibody, protein, or oligonucleotide destined for immobilization onto the surface. 

Scheme 1 Examples of Chemoselective Ligation Strategies Resulting in the Formation of Nonpeptidic Bonds (a) Hydrazone, (b) Oxime, (c) Thiazolidine...

To 1 volume of the hydrazone mixture there was added, with mixing, 2 volumes of 0.6 M NaBH3CN in 0.1 M NaOAc/MeCN (1 1) the apparent pH was previously adjusted (glass electrode) to 4.6 with glacial AcOH. The apparent pH was readjusted to an apparent value of 4.6 with glacial AcOH, the soln incubated for 4d at rt in the dark, and the reduced hydrazone finally isolated by HPLC. The same conditions could be used for both the ligation steps shown in Scheme 2. 

Comparative Study of Imine Ligation Oxime, Hydrazone, and... 

Ligation Conditions of Peptide Dendrimer Formation through Oxime, Hydrazone, and Thiazolidine Ligations To Form 64, 66, or 70 [85 ... 

Hydrazone Ligation of (HCOCO)4-K2K 20a and (HCOCO) -di-K2K Dendrons 22a with 4-Hy-drazinobenzoyl-SRlO 67 To Give 68 [891... 

Finally, several groups have reported irreversible chemistries that work selectively in the presence of proteins or even whole cells. These include the Huisgen cycloaddition recently improved by Sharpless and others,1146-481 chemical ligation,1491 the formation of oximes or hydrazones and the Staudinger ligation.1501... 

Azoles can be produced from products of palladium-catalyzed hydrazone arylation and can serve as substrates for arylation reactions to produce N-aryl azoles. The Fischer indole synthesis uses N-ary I hydrazones, and these hydrazones can be generated by palladium-catalyzed chemistry. Benzophenone hydrazone was found by both the Yale and MIT groups to be a particularly effective substrate for palladium-catalyzed reactions, as summarized in Eq. (24) [140,141]. Reactions of benzophenone hydrazone with either aryl bromides or iodides occur in high yields using either DPPF- or BINAP-ligated palladium. These reactions are general and occur with electron-rich, electron-poor, hindered or unhindered aryl halides. The products of these reactions can be converted to hydrazones that bear enolizable hydrogens and are suitable for indole synthesis in the presence of acid and ketone [140]. 

To date, four bioorthogonal reactions have been used to label glycans on cells and in lysates hydrazone/oxime formation with ketones, thiol alkylation with maleimides, Staudinger ligation of azides with triaryl phosphines, and copper-catalyzed or strain-promoted [3+2] cycloadditions of alkynes and azides (Figure 5) (25, 26, 32, 34, 35). While each reaction has been used extensively, most recent applications have employed azido- or alkynyl-sugars due to their superior metabolic incorporation and efficient ligations. 

Figure 5. Bioorthogonal reactions on sugars, A. Ketones react with hydrazides to give hydrazones. B. Thiols undergo Michael Reaction with maleimides. C, Azides undergo Staudinger ligation with phosphines or D. strain-promoted or copper catalyzed [3 2] cycloaddition with all nes. E. Alkynes undergo copper-catalyzed [3- 2] cycloaddition with azides.

The reactions presented above are those most commonly associated with the term chemoselective ligation although there are more reactions which fall into this class. A combination of two orthogonal ligation reactions, hydrazone formation from a hydrazine and an aldehyde (often a glyoxal adduct), and thioether ligation (by substitution of a chloroacetyl group by an in situ formed thiol) in a one-pot procedure have successfully been carried out [203],... 

Fig. 10.2-3 Hydrazone ligation of unprotected peptides in aqueous solution.

Examples of efficient ligation methods for creating artifically linked proteins or peptides include formation of thioesters by reaction of a thioacid and a bromoacetamide [119], formation of pseudoproline residues from an aldehyde and Cys or Thr [122,124], formation of an oxime from an amino-oxy function and an aldehyde or ketone [120,121], and formation of a hydrazone from a hydrazide and an aldehyde [121,125,126]. Several of the preceding methods may be used in series because of different reactivities. 

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